As the list of expressed human genes expands, a major scientific and medical challenge is to understand the molecular events that drive normal tissue morphogenesis and the progression of pathologic lesions in actual tissue. With refinements in PCR, microhybridization arrays and mutation screening, DNA or mRNA can be extracted from tissue biopsies and analyzed with a parallel panel of hundreds or even thousands of genetic markers. Since complex tissues consist of multiple cells types biochemically and physically affected by surrounding cells and molecular environments, the task of analyzing critical gene expression patterns in development, normal function, and disease progression depends on the identification and extraction of specific cells from their complex tissue milieu. Laser capture microdissection (LCM) has been developed by our lab in collaboration with NCI and ORS and then through a CRADA with Arcturus to provide a rapid, reliable method to procure pure populations of specified cells from specific microscopic regions of tissue sections for subsequent quantitative, multiplex molecular analysis. In the last year we have further refined LCM instrumentation for targeting of single cells and concentrating rare cells onto the transfer surface and explored new laser ablative technologies for isolating single cells from tissue sections on polymer thin films. A detailed physical understanding of the polymer activation and capture process within both systems has lead to new approaches of "noncontact" LCM suitable for isolating rare cell populations in both tissue sections and cytology. We have demonstrated reproducible single cell capture and analysis of specific genetic alterations by PCR and sequencing. We have improved laser microdissection methods for quantitative capture and extraction of DNA, protein and mRNA, most notably full length mRNA in LCM samples used to generate high quality, complex cDNA libraries of homogeneous populations of cells in developing embryos and in animal models of disease. We have begun global analyses of gene expression using microarray hybridization and have examined characteristic statistical patterns of gene expression. Through a commercial CRADA and hosting a series of international conferences on Laser Microdissection, we have spurred the rapid dissemination of a commercial technology into widespread use in ~600 research labs around the world. LCM-based molecular analysis of histopathological lesions can be applied to any disease process that is accessible through tissue sampling. The fluctuation of expressed genes or alterations in the cellular DNA which correlate with a particular disease stage can be compared within or among individual patients. Such a fingerprint of gene expression patterns may provide crucial clues for etiology, and may ultimately contribute to diagnostic decisions and therapies tailored to the individual patient. In collaborations with other labs on animal models, we are currently studying gene expression patterns associated with normal development of the testes and thyroid as well as the induction and progression of plasmacytomas induced by chronic inflammation. Our lab is developing robust statistical tools to be applied to such multidimensional datasets in order to identify critical genes, pathways, and the complex integration of transcription, translation, and post-translational modification of large number of genes that characterize normal cellular function and the deviations characteristic of specific pathologies. Sets of macromolecules found to be uniquely associated with a defined pathological lesion in clinical studies may serve diagnostic imaging markers in screening of populations at risk and for evaluating response to therapy designed to prevent progression. We are investigating the role of laser microdissection both in clinical screening and response to early intervention designed to prevent progression.